Abstract: Amongst the roughly 4 million Americans infected with the Hepatitis C Virus (HCV), chronic alcohol use has long been known to dramatically accelerate the progression of liver disease. Recent analysis has shown that long-term alcohol exposure increases the risk of death from HCV more than 8-fold over viral infection alone. Despite its importance as one of the strongest independent predictors of both cirrhosis and death, very little is known about the nature of the interaction between alcohol and HCV pathogenesis. Previous work from the laboratories collaborating on this application has shown that ethanol exposure impairs protein methylation in liver cells and reduces IFN-induced signaling along the JAK-STAT1 pathway (pathway that limits viral replication). Others have demonstrated that hypomethylation leads to activation of HCV NS3/4 protease, which may potentially block host innate antiviral immunity through NS3/4a-mediated cleavage of the adaptor protein, MAVS and that hypomethylation of STAT1 causes suppression of its attachment to DNA. This allows formulating our central hypothesis that ethanol exposure exacerbates HCV infection by impairing interferon signaling in HCV-infected and uninfected hepatocytes via dysregulation of methylation reactions. To test the hypothesis, we propose three Specific Aims to perform detailed examination of the effects of ethanol exposure/impaired methylation and HCV-infection on human innate hepatocyte immunity utilizing both in vitro and in vivo models. In Specific Aim 1, we will investigate the in vitro effects of ethanol on HCV replication and interferon signaling in HCV-infected/ expressing cell lines and human hepatocytes. In Specific Aim 2, we will study the in vivo effects of ethanol and impaired methylation on HCV load and interferon signaling in mice (scid-Alb/uPA) with chimeric human livers. In Specific Aim 3 we will assess whether the correction of ethanol- induced methylation defects by the pro-methylating agent, betaine, restores interferon signaling and improves HCV clearance in infected mice treated with IFN2b. The results of these studies will aid in the determination of the central role for ethanol-impaired methylation in IFN signaling for suppression of host defense, viral replication and pathogenesis. In addition, we will also obtain data in support of the use of pro-methylation agents in the treatment of HCV-infected patients with chronic alcohol use. These findings would ultimately lead to a vertical step in the field through direct extension to a clinical trial using this novel approach with the long-term goa of reducing the excessive morbidity and mortality that HCV infection causes in patients with chronic alcohol use.